Technical Development of Photoprobes for Physiology and Neuroscience

We design, synthesize, and develop biological applications for, molecular optical probes, or "photoprobes." Photoprobes fall into two classes: fluorescent indicators and "caged" molecules. A fluorescent indicator is simply a molecule that responds to a change in its physical or chemical environment by displaying changes in its fluorescence properties. Fluorescent probes currently under development are: 1) a calcium indicator that has very low diffusional mobility and thus should allow us to observe fast, localized changes in [Ca2+] that occur in subcellular domains; and 2) a membrane potential indicator that allows the voltage across the cell membrane to be monitored without having to use electrodes or electronic amplifiers. Such an indicator would immediately allow us to track the electrical behavior of ensembles of nerve cells that are linked in a neural circuit. A caged probe is an inert but photosensitive precursor molecule that can be photolyzed by a pulse of light to generate a biologically active effector molecule in situ. Caged probes developed in this lab now allow us to use light to modulate SR/ER Ca pumps, and to release small signaling molecules (e.g. CO), inorganic ions (e.g. Ca2+ and H+) that have cellular signaling functions, as well as neurotransmitters such as glutamate and g-aminobutyric acid (GABA). The coupling of such novel reagents with newly developed techniques for delivering light pulses allows the experimentalist to manipulate biological processes in living specimens with unprecedented spatial and temporal control.

Biology

Calcium Regulation of Neuronal Excitability. Following stimulation by action potentials, a slowly-developing and long-lasting hyperpolarization (AHPslow) is observed in many types of neurons. Such AHPslow's represent a fundamental mechanism by which neuronal excitability can be modulated. A robust AHPslow occurs in vagal afferent (nodose) neurons of the adult rabbit and ferret. Our studies have revealed that generation of the AHPslow is obligatorily dependent on Ca2+ from intracellular stores whose release is triggered by action-potential-evoked Ca2+ influx, in a process of Ca2+-induced Ca2+ release (CICR). The nodose neuron is thus the first instance, apart from cardiac myocytes, where CICR has been demonstrated to mediate a physiologically important process. Current research focuses on elucidating the molecular signaling steps that allow Ca2+ to turn on the AHPslow. (Collaboration with Dr. Daniel Weinreich.)

Probing Synaptic Integration by Photostimulation

A single neuron in the central nervous system may receive inputs from thousands of neighboring neurons through as many as 104 synapses located on its highly-branched dendritic tree. From the myriads of spatially and temporally distinct synaptic inputs, a single sequence of discrete electrical pulses is generated by the neuron as output. The procedure by which the dendritic tree processes diverse inputs separated in time and space is termed dendritic integration. By using light pulses to generate neurotransmitter (e.g. from caged glutamate) at different times and at different locations on the dendritic tree, while the electrical response of the neuron is monitored, we can systematically document how the neuron responds to stimulatory inputs that have well-defined spatial and temporal relationships. In this way, information processing algorithms that are "hard-wired" into the dendritic tree can be deciphered. (Collaboration with Dr. Cha-Min Tang.)